CELL STRESS FOR PATHOGEN SUCCESS IN TICKS: ATF6 AND NRF2 REGULATORY NETWORKS SUPPORT ANAPLASMA PHAGOCYTOPHILUM

Kaylee Ann Vosbigian

Incidence of tick-borne disease is on the rise. How tick-borne pathogens interact with their hosts has been studied mostly in vertebrates where disease is observed. Much less is known about how pathogens interact within the tick. In order to develop preventative control measures for tick-borne diseases, we must first understand how ticks acquire, harbor and transmit pathogens. The North American deer tick, Ixodes scapularis, can transmit seven known pathogens affecting human health, including Anaplasma phagocytophilum (causative agent of anaplasmosis) and Borrelia burgdorferi (causative agent of Lyme disease). Stress imparted on ticks by these pathogens activates the unfolded protein response (UPR), a conserved cellular stress response involved in maintaining homeostasis. Here, we investigate corresponding regulatory networks of the UPR and their effects on infection in ticks.We show that the UPR receptor, ATF6, supports pathogen survival in ticks. We investigated the Ixodes ATF6 transcriptional network by developing a custom R script to query promoter sequences within the tick genome. This revealed stomatin, which is involved in lipid homeostasis and vesical transport within the cell. We experimentally validated stomatin as being ATF6 regulated through luciferase reporter assays, pharmacological inhibitors and repression of transcripts through RNAi. We demonstrate that stomatin supports Anaplasma colonization in ticks and interrupts cholesterol dynamics. Additionally, decreasing stomatin restricts cholesterol availability to Anaplasma inhibiting growth and survival. Furthermore, we developed a custom web-based transcription factor binding search tool termed “ArthroQuest”, which revealed that ATF6-regulation of stomatin could be unique to blood-feeding arthropods. In addition to UPR activation, pathogens also elicit antioxidant responses. The antioxidant response protects tick cells from damage from reactive oxygen species, inadvertently supporting pathogens. The master regulator of antioxidant responses, Nrf2, is also activated downstream of the UPR receptor, PERK. We previously found that Nrf2 is activated during Anaplasma infection in ticks. To investigate the Nrf2 regulatory network we scanned promoter sequences for Nrf2 binding sites in ticks. We discovered nucleoporin 214 as putatively regulated by Nrf2 and upregulated during tick infection. We validated Nrf2 regulation of nup214 through Luciferase reporter assays and RNAi transcriptional knockdowns. We demonstrated that Nup214 supports Anaplasma infection in ticks and amplifies the antioxidant response by maintaining Nrf2 nuclear localization. Altogether, we show how the tick’s UPR plays a protective role during Anaplasma infection in ticks. This work elucidates mechanisms unique to tick infection which differ from that in mammals, which highlights the importance of studying host-pathogen dynamics in non-model organisms. Additionally, this research has also advanced tools for vector-borne disease investigators and demonstrates the importance of combining traditional benchwork with silico methods.

CELL STRESS FOR PATHOGEN SUCCESS IN TICKS: ATF6 AND NRF2 REGULATORY NETWORKS SUPPORT ANAPLASMA PHAGOCYTOPHILUM

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